Original Contributions |
From the Department of Pathology, University of Washington (D.W.C., S.M.S.), and ZymoGenetics (C.E.H.), Seattle, Wash.
Correspondence to Dr Charles E. Hart, ZymoGenetics, 1201 Eastlake Ave East, Seattle, WA 98102. E-mail HARTC{at}zgi.com
| Abstract |
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Key Words: factor VII tissue factor fibrin arteries tunica intima
| Introduction |
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The term remodeling has been used to describe different processes in the literature. For the present study, we have adopted the definition of Mulvaney,5 who has defined eutrophic remodeling as the process of structural change in a blood vessel with no net growth in the wall constituents. Eutrophic remodeling can occur as either inward remodeling (loss of lumen) or outward remodeling (gain in luminal area); however, the basic tenet is that the relaxed vessel has undergone a structural rearrangement that does not involve either gain or loss of vessel wall mass. By this definition, the more general term, remodeling, refers to any structural changes in a vessel that alter the lumen of a relaxed vessel when measured under standard intravascular pressure.
The eutrophic remodeling concept may be even more important in
atherosclerotic progression than in restenosis. In 1987, Glagov
et al10 provided evidence that coronary
arteries adapt to large gains in intimal mass by increasing their
external circumference, a process termed compensatory enlargement. This
adaptation preserves luminal area until a critical limit of intimal
mass, representing
40% of preadapted luminal area, is
reached. The concept of compensatory enlargement has been confirmed in
necropsy studies of both primate and human coronary
vessels11 as well as in a case study using
sequential intravascular ultrasound.12
Compensatory enlargement of iliac arteries has also been observed from
2 to 4 weeks after angioplasty in a primate model of
atherosclerosis.13
Stenosis may therefore represent a failure in
compensatory enlargement. Thus, we not only lack a complete explanation
for the loss of lumen after angioplasty, but we also have no
satisfactory hypothesis for the progressive stenosis that
occurs even in atherosclerotic vessels in humans.
Animal studies described as models of restenosis also illustrate the importance of distinguishing neointimal hyperplasia, in response to various procedures, from luminal narrowing. A key study by Langille and O'Donnell14 showed the role of the endothelium in flow-dependent remodeling of blood vessels to smaller luminal diameters. In this model, denudation injury, a method commonly used to produce intimal hyperplasia, eliminated luminal loss in response to decreased blood flow. Recently, several small animal models have shown that intimal growth can only partially account for loss of gain after angioplasty.15 16 17 It is probably correct at this point to say that although intimal mass, especially atherosclerotic mass, may contribute to loss of lumen, loss of luminal area must also depend on loss of the intrinsic ability to accommodate an as-yet-unidentified pathological process that promotes vascular narrowing in injured vessels.
The present study attempts to explore one possible mechanism for pathological narrowing after injury. We hypothesized that luminal loss is at least partially dependent on the procoagulant property of the neointima,18 19 a property also described in advanced atherosclerotic lesions.20 In the rabbit, previous studies have shown that single injuries produce little evidence of fibrin formation, yet sequential injuries on a preformed neointima produce extensive mural fibrin deposition.21 22 23 We have therefore chosen sequential injury to the rabbit abdominal aorta combined with the use of a specific tissue factor inhibitor (FVIIai) to examine the role of the extrinsic pathway of coagulation in blood vessel remodeling. In order to characterize the remodeling process, we have directly measured both luminal loss, which we have quantified by vascular casting, and intimal hyperplasia.
| Materials and Methods |
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Rabbits in each group were killed either at 4 hours (n=4), 72 hours (n=6), or 3 weeks (n=6) after the final injury. For the 3-week time point, rabbits were perfusion-cast to provide accurate luminal areas. For all prior time points, the vessels were perfusion-fixed. The 4-hour time points were used to examine mural and intramural fibrin deposition by scanning electron microscopy and immunohistochemistry. For the 72-hour time point, rabbits were infused with BrdU before they were killed for study, and the tissue was used to examine cell proliferation.
To determine whether the luminal loss was progressive, 7 more rabbits were cast 6 weeks after a second balloon denudation injury, and 4 more rabbits were cast at 6 weeks after a single injury. To obtain baseline values, the vessels from 5 rabbits (age- and weight-matched to the 6-week time point) were cast without any vascular injury. An additional 8 rabbits (4 FVIIai-treated and 4 vehicle control rabbits) were killed at 4 hours and 10 days after sequential injury in order to obtain fresh frozen tissue for immunohistochemical study.
Balloon Denudation
Rabbits were anesthetized with intramuscular and
intravenous injections of ketamine (Ketaject,
Phoenix) and xylazine (Rompun, Miles Inc). A 4F Fogarty embolectomy
catheter (Baxter) was inserted via a left femoral arteriotomy and
passed 20 cm into the abdominal aorta. The catheter was inflated and
drawn back to the iliac bifurcation. Catheter insertion and inflation
were repeated for three passes, after which the catheter was removed,
the femoral artery was tied, and the incision was closed. The analgesic
buprenorphine (0.06 mg, Buprenex, Reckitt and Coleman) and the
antibiotic penicillin G (60 000 U, Phoenix) were applied
intramuscularly.
For group 2 rabbits, a second balloon denudation was performed 3 weeks after the first procedure. The procedure was identical except that the right femoral artery was used for access and a 3F Fogarty balloon used for denudation. The flow reduction produced by occlusion of both femoral arteries was measured in two rabbits. A transonic flow probe (Transonic Systems Inc) was placed around the central abdominal aorta, and mean flow was repeatedly recorded before and after clamping of the femoral arteries. Clamping produced only minor changes in flow rate, with a mean flow reduction of 9%.
Plasma samples (in 10% sodium citrate, Sigma Chemical Co) were collected at three time points: (1) before balloon denudation (and before administration of FVIIai or vehicle), (2) immediately after ballooning, and (3) 3 days after the final balloon denudation. For rabbits surviving >3 days, a final plasma sample was drawn before they were killed for study. Plasma samples were stored at -80°C for later analysis.
Drug Preparation and Administration
FVIIai was generated from recombinant wild-type human FVIIa as
previously described.24 FVIIai produced a
dose-dependent prolongation of coagulation times in a standard partial
thromboplastin coagulation assay with normal rabbit plasma and rabbit
thromboplastin.
The FVIIai dosage was based on the short half-life (
30 minutes in
rabbits) and a requirement for high circulating levels of FVIIai during
the perioperative period. Intravenous bolus
injections of FVIIai (6 mg, in Tris-buffered saline, pH 7.4) were
administered 30 minutes before balloon denudation, immediately after
catheter insertion, and 2, 4, and 8 hours after denudation. For
prolonged administration, two osmotic minipumps (model 2mL1, Alzet)
were loaded with 2 mL of FVIIai (3 mg/mL) and implanted
intraperitoneally. These pumps supplied FVIIai at
60 µg/h over the 1-week period following injury. Control rabbits were
given bolus doses of vehicle (Tris-buffered saline, pH 7.4) and were
implanted with two osmotic minipumps loaded with vehicle. Animals in
the sequential injury group were given the same drug regimen at the
time of the second injury.
For cell replication studies, BrdU (Sigma) was administered intraperitoneally in a loading dose (40 mg in 2 mL H2O) and continuously for 24 hours with an osmotic minipump (model 2001, Alzet) delivering 1.1 mg of BrdU/h. Rabbits were killed 24 hours after initial administration.
Tissue Preparation
Rabbits were heparinized (1000 IU IV, Elkins-Sinn) and killed by
anesthetic overdose. The thoracic aorta was cannulated, and the
arterial system was flushed with 120 mL of lactated
Ringer's solution. For the 4-hour and 3-day time points, the aortas
were perfusion-fixed for 20 minutes at 100 mm Hg with 3%
paraformaldehyde (phosphate-buffered, pH 7.4; time and
pressure were optimized to attain adequate fixation while limiting
extensive use of fixative). The abdominal aorta from the renal to the
iliac bifurcation was resected and fixed overnight in 3%
phosphate-buffered paraformaldehyde. To obtain luminal
morphometry, aortas at the 3- and 6-week time points and the uninjured
control aortas were cast with Batson's compound (Batson's No. 17,
Polysciences) infused at an infusion pressure of 120 mm Hg, which
was maintained until the compound had set. The aorta and cast were
resected and fixed ex vivo for 1 week in 3% phosphate-buffered
paraformaldehyde.
Three portions of each abdominal aorta (1 cm in length) were taken
between lumbar vessels beginning with the intralumbar region
2 cm
from the iliac bifurcation and progressing proximally (see Figure 2
). For aortic casts, the 1-cm segments
were cut from the cast with a hand drill with a carbide blade. Portions
of the aorta were then gently pulled from the cast. The cast was
preserved for determination of luminal diameter and area, and tissue
segments were paraffin-embedded. For scanning electron microscopy,
sections of aorta were postfixed in 1% glutaraldehyde
(phosphate-buffered, pH 7.4), pinned open longitudinally, fixed in
osmium tetroxide, critical pointdried, and sputter-coated.
|
Histomorphometry
Cross sections of abdominal aorta were stained with the
following: (1) Verhoeff's elastic tissue stain to differentiate the
elastic lamellae, (2) hematoxylin and eosin, and (3) Gomori's
trichrome connective tissue stain.18 Because of
the large size of the aortic cross sections, a single low-power
digitization proved to be less reproducible than a sampling technique
in which cross-sectional areas were determined from multiple fields of
x200 magnification. For each Verhoeff's stained cross section, eight
randomly chosen fields were digitized. With this sampling technique,
50% of the entire cross-sectional area was measured. Optimas image
analysis software (Bioscan Inc) was used to measure intimal and
medial areas as well as the linear portion of the circumference for
each sample field. The latter measurement was divided into the area
calculation for each field, and the mean intimal area and medial
areatofield length ratios were calculated. The vessel circumference
was measured under low power, and this measurement was multiplied by
the areatofield length ratio to calculate the total cross-sectional
area for each vessel cross section. For measurement of EEL, area
low-power magnification was used to digitize the perimeter length of
the EEL. Because of possible distortion of the lumen in tissue
processing, a cylindrical assumption was used to calculate areas from
perimeter length.
For each abdominal aorta, sections from the proximal, central, and distal regions were analyzed. The mean values for each animal were used in the statistical analysis.
Immunohistochemistry and Fluorescent Staining
Immunohistochemistry was performed with the avidin biotin
complex system (Vectastain, Vector Laboratories). Paraffin sections
were cleared, rehydrated, and washed in PBS (pH 7.4). A 10-minute
pepsin (Biomeda) digest at 37°C followed by a PBS wash was performed;
the sections were then blocked with 2% horse serum. Sections were
incubated with primary antibody for 1 hour at 37°C, washed, and
incubated with a biotinylated secondary antibody for 1 hour at 37°C.
After the sections were washed, the streptavidinhorseradish
peroxidase conjugate was applied, and the sections were stained with
diaminobenzidine. For each run, a negative control was processed by
deleting the primary antibody and applying an isotype-specific control
antibody (Zymed).
Frozen sections were thawed, washed in PBS, dehydrated in ethanol, and acetone-fixed for 10 minutes at 4°C. Endogenous peroxide was blocked with a 5-minute incubation with 1 part hydrogen peroxide (30%) in 9 parts methanol. Sections were then rehydrated to PBS. Tissue blocking and antibody staining proceeded as described for paraffin sections.
The primary monoclonal antibodies used were specific to smooth muscle
-actin (Sigma), fibrin (No. 350, American Diagnostica
Inc), and BrdU-labeled DNA (Biomeda). Before BrdU staining, DNA was
denatured by application of 2N HCl for 20 minutes. To characterize the
fibrin-specific antibody, rabbit plasma was clotted with bovine
thrombin and embedded in gelatin. Citrated plasma was also embedded in
gelatin. Frozen sections were then fixed in 3% phosphate-buffered
paraformaldehyde and stained. The antibody was
demonstrated to be plasma clot specific in the rabbit.
FVIIai was conjugated with the fluorescent dye Oregon Green 514 by use of a labeling kit (Molecular Probes). FVIIai was dialyzed to PBS (pH 7.4), and 200 µL of 2 mg/mL FVIIai was reacted with 20 µL sodium bicarbonate (pH 8.3) and 7.8 µL dye solution (10 mg/mL in dimethyl sulfoxide) for 90 minutes at room temperature. The reaction was terminated by the addition of excess hydroxylamine, and labeled FVIIai (OG-FVIIai) was purified with a spin column.
For OG-FVIIai staining, paraffin sections were cleared, rehydrated, and washed in Tris-buffered saline (pH 7.4). OG-FVIIai was diluted 1:20 in Tris-buffered saline containing 20 mmol/L CaCl2 and applied to the sections for 20 minutes. The sections were then washed for 10 minutes with three changes of Tris-buffered saline. To control for specificity, 20x excess unconjugated FVIIai was used to compete off OG-FVIIai. All sections were mounted with Vectashield (Vector Laboratories), and micrographs were immediately taken on an Olympus BH-2 fluorescent microscope.
In Vitro Plasma Assays
In order to measure FVIIai serum levels, a monoclonal-polyclonal
sandwich ELISA was used. An anti-human factor VIIa monoclonal antibody
(courtesy of Walt Kisiel, University of New Mexico, Albuquerque) was
plated onto 96-well microtiter dishes (Maxisorp, Nunc) by adding a
concentration of 2.0 µg/mL and a volume of 100 µL to each well, and
the plates were incubated overnight at 4°C. The plates were then
washed in washing buffer (PBS and 0.05% Tween 20, pH 7.4) and
subsequently blocked for 2 hours at 37°C with blocking buffer (PBS,
0.05% Tween 20, and 1% BSA, pH 7.4). Dilutions of FVIIai (20 to 0.027
ng/mL) or sample plasma were made in blocking buffer, 100 µL of test
sample or plasma was added to the plates, and the plates were incubated
for 1 hour at 37°C and subsequently washed. A rabbit anti-human FVIIa
polyclonal antibody (courtesy of W. Kisiel), diluted 1:1000 in blocking
buffer, was added to the wells, and the plates were incubated for 1
hour at 37°C. Goat anti-rabbit IgG conjugated with horseradish
peroxidase (Sigma) was then added, and the plates were incubated for 1
hour at 37°C. The plates were subsequently washed, and the substrate
(O-phenylenediamine dihydrochloride) was
added. The absorbance was read at 490
, and FVIIai concentrations
were determined from a standard curve.
Plasma was also examined for prolongation of partial thromboplastin time in a standard assay with rabbit thromboplastin (Sigma). Duplicate cuvettes containing 50 µL of rabbit plasma and 150 µL of Tris-buffered saline (10 mmol/L Tris and 0.15 mol/L NaCl) were loaded into an automatic coagulation timer (Electra 800, Medical Laboratory Automation Inc). Recalcification was accomplished with a 1:3 dilution of thromboplastin in Tris-buffered saline with 25 mmol/L CaCl2, and time to clot was recorded. Plasma levels of FVIIa were determined for control samples with a modified coagulation time assay specific for FVIIa, as described by Wildgoose et al.25
Statistical Analysis
Statview software (Abacus Concepts Inc) was used for statistical
analysis. Comparisons of drug-treated groups with control
groups at each time point were made with the Student t test.
An ANOVA and Fisher protected least significant differences were used
to compare the effect of single and double injury at the various time
points. Paired t tests with a Bonferroni correction were
used to compare individual plasma samples over the time course of the
experiment. All values in the text are quoted as mean±SEM.
| Results |
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Treatment with FVIIai had no statistically significant effect on the
level of medial cell replication 3 days after injury (200±21
BrdU-labeled cells per cross section, Table 1
). Similarly, FVIIai
treatment had no effect on intimal growth or on luminal area at 3 weeks
after balloon injury (Figure 4
).
|
Sequential Injury
At 4 hours after sequential injury, histological
cross sections displayed an intact neointima. The mean
number of BrdU-positive cells (per cross section) in the media 72 hours
after the second injury was low (33±20, representing a
proliferation index of 0.79%), with cell proliferation largely
confined to the preformed intima with a mean of 178±40 cells labeled
(7.6% proliferation index) in the intima of vessel cross sections.
Total cell proliferation per vessel cross section was similar to the
single injury when both intimal and medial cells were considered
(211±60 for sequential injury versus 204±32 for a single injury).
Proliferation indices in the intima 72 hours after the sequential
injury were similar to indices in the media after a single injury (see
Table 1
).
Sequential injury produced a progressive loss of luminal area not seen
in animals injured only once (Figure 3
). At 3 weeks after sequential
injury, the mean luminal area, as determined from direct measurements
of aortic casts, was 80% of that from vessels of uninjured animals
(12.3±0.5 mm2 [uninjured] versus
9.8±0.6 mm2 [injured],
P=0.03). At 6 weeks after the second injury, mean luminal
diameter had dropped to 55% of the uninjured vessel diameter
(12.3±0.5 mm2 [uninjured] versus
6.8±0.5 mm2 [uninjured],
P<0.001).
FVIIai treatment eliminated the luminal narrowing observed at 3 weeks
after the second injury (mean luminal cross-sectional areas from casts
were 14.3±1.4 mm2 for FVIIai-treated
vessels versus 9.8±0.6 mm2 for control
vessels, P=0.03 by t test) (Figure 4
). The
greatest loss in luminal area in control vessels was seen in the distal
abdominal aorta (2 cm proximal to the iliac bifurcation), and the
effect of FVIIai treatment was greatest in this location (data not
shown). Measurements of EEL area from histological
cross sections were also made for analysis of total vessel
area. The EEL measurements, 13.6±1.2 and 10.0±0.7
mm2 for FVIIai-treated and control vessels,
respectively, also showed an increased total vessel area in
FVIIai-treated animals (P=0.03) similar to the direct
luminal measurements made from casts.
Luminal narrowing could not be attributed to gain in intimal area
because there was no difference between intimal area in control and
treated vessels (Figure 4
). FVIIai treatment also had no significant
effect on cell replication rates, as measured by BrdU staining after 3
days (Table 1
). Vessel wall structure
examined by light microscopy revealed no morphologically distinct
differences between FVIIai-treated and untreated vessels.
Fibrin Deposition After Single and Sequential Injuries
No fibrin deposition was observed on aortic cross sections taken 4
or 72 hours after a single balloon injury, and immunohistochemistry
with a fibrin-specific antibody displayed only background staining
(data not shown). However, in control treated animals receiving
sequential injury, extensive fibrin deposition was observed 4 hours
after injury. One third of the vehicle control rabbits had mural
thrombus visible on gross inspection. All sequential injuries had
morphologically visible fibrin on the luminal surface, as evidenced by
light microscopy of Gomori's stained sections and scanning electron
microscopy (Figure 5a
). In sequential
injury, the extent of fibrin deposition on the luminal surface was
variable, ranging from scattered deposits to extensive coverage of
the injured arterial surface. Immunohistochemical staining
for fibrin revealed extensive mural and intramural fibrin deposition in
control aortas examined 4 hours after a second injury. Dense fibrin
staining was observed in the preformed intima (Figure 6a
). This dense staining did not extend
past the internal elastic lamina; however, scattered regions of
staining for fibrin did appear in both the media and adventitia of
control vessels. At 10 days after the second injury, fibrin staining
was still observed in the intima, with regions of intense staining near
the luminal surface and the internal elastic lamina (Figure 6c
).
|
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Mural and intramural fibrin deposition was largely eliminated in
FVIIai-treated animals examined 4 hours after the second balloon
injury. In order to assess the extent of fibrin deposition within the
vessel wall and on the luminal surface, three distinct methods were
used. First, extensive areas of the vessels were examined for mural
thrombus with Gomori's stained sections. The extent of mural fibrin
deposition was assessed semiquantitatively in a double-blind manner and
scored from 0 to 4. Sections from rabbits receiving vehicle scored
significantly higher (mean score of 3) than did sections from
FVIIai-treated rabbits (mean score of 0). Second, the surface of
vessels 4 hours after sequential injury were examined by scanning
electron microscopy. Fibrin and platelet-rich thrombi were
frequently seen in scattered regions of the luminal surface of vessels
from rabbits treated with vehicle (Figure 5a
). In FVIIai-treated
vessels, fibrin was not observed on the luminal surface of specimens
examined by scanning electron microscopy (Figure 5b
). However, limited
platelet deposition was observed on the luminal surface of these
vessels. Third, the extent of intramural fibrin deposition was assessed
by immunohistochemical staining of aortic cross sections with a
fibrin-specific antibody. FVIIai-treated vessels displayed very limited
staining within the vessel wall at both 4 hours and 10 days after the
sequential injury (Figure 6b
and 6d
). In contrast, large dense patches
of staining were observed within the intima of vessels from vehicle
control rabbits.
Localization of Tissue Factor
To examine the spacial localization of tissue factor within the
vessel wall, tissue cross sections were stained with
fluorescent FVIIai (OG-FVIIai). Uninjured vessels displayed
intense staining only in the adventitia (not shown). At 3 days after a
single injury, diffuse staining developed throughout the media and
developing intima (Figure 7a
). Three
weeks after a single balloon denudation injury, OG-FVIIai displayed
intense staining in the neointima and diffuse staining in
the media (Figure 7b
). At 4 hours after sequential injury, intense
staining in the intima could still be observed in vessels of vehicle
control rabbits (Figure 7c
). The intense intimal staining with
OG-FVIIai occurred in the same regions as the antifibrin staining after
sequential injury in control treated animals (Figure 6a
). Rabbits
treated with FVIIai displayed very little OG-FVIIai staining (Figure 7d
), indicating that the tissue factor in these sections was not
available for binding. OG-FVIIai staining could also be eliminated by
coincubation with 10-fold excess unlabeled FVIIai (not shown).
|
Plasma Coagulation Factors
Intravenous injections of FVIIai at the time of the
operation produced high circulating levels immediately after the
operation, as measured by ELISA on plasma samples (Table 2
). Lower
circulating levels were maintained for 1 week after the operation by
intraperitoneal delivery from osmotic pumps.
Consistent with the ELISA results, plasma partial
thromboplastin clotting times in the immediate postoperative period
were significantly elevated. However, by 3 days after the operation,
despite the low levels of circulating FVIIai, partial thromboplastin
times were slightly depressed in both treated and control animals.
There were no statistical differences for plasma levels of FVIIai or
for partial thromboplastin times in rabbits in the single or sequential
injury group. Values for the sequential injury group are summarized in
Table 2
.
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| Discussion |
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Activation of the coagulation cascade has been implicated in both smooth muscle cell mitogenesis and chemotaxis27 28 29 30 ; however, in terms of neointimal formation, results have not been consistent between animal models. Short-term application of the direct thrombin inhibitor, hirudin, decreases neointimal area after injury of the rabbit carotid yet has no effect in the rat or minipig.26 Jang et al24 demonstrated that both FVIIai and tissue factor pathway inhibitor decreased the extent of stenosis and intimal area (measured at stenotic regions) after sequential injury to the iliac artery in hypercholesteremic rabbits, whereas hirudin and tick anticoagulant peptide had no significant effect. Our studies using a sequential injury to the aorta in normocholesterolemic rabbits showed that inhibition of the extrinsic pathway with FVIIai had no significant impact on either neointimal area or smooth muscle cell proliferation (measured 3 days after injury), indicating that coagulation is not the major stimulus for proliferation in this model. These results are consistent with the findings of Ragosta et al,31 who have shown in a balloon angioplasty model in the hypercholesterolemic rabbit that hirudin treatment was not associated with decreased early smooth muscle cell proliferation despite a significant reduction in plaque size.
As with any animal model, the results from the present study may not be directly predictive of remodeling in diseased human coronary arteries. Possible limitations of the model described herein involve the use of the rabbit abdominal aorta, an elastic vessel that is structurally distinct, with no attempt to develop focal stenoses or plaques. Rather, we have concentrated on the remodeling of the entire vessel in response to sequential mechanical injury. In rabbit models involving hypercholesteremia, focal intimal lesions that are rich in inflammatory infiltrates develop. Remodeling in these hypercholesteremic vessels is clearly multifactorial and involves extensive intimal thickening. The strength of the model that we describe in the present study is that pathological eutrophic remodeling to smaller luminal diameters can occur in vessels with no extensive intimal thickening or inflammation, indicating a distinct mechanism of arterial remodeling. We believe, but have not proven, that this distinct eutrophic remodeling process is one component of vessel remodeling that can occur along with other remodeling processes (such as intimal growth) in mechanically injured severely atherosclerotic arteries.
In the model described in the present study, loss of lumen was not
driven by an enlarging neointima. However, there was a weak
inverse correlation between luminal area and intimal area in the
untreated group (r2=0.412,
P=0.04 for correlation). This correlation suggests that
properties of the neointima are involved in the inward
eutrophic remodeling process. One possibility is that a larger
neointima produces more extensive fibrin deposition and
that the fibrin is important for the eutrophic remodeling response.
There are several possible mechanisms by which coagulation may be
involved. The fibrin that we observed could lead to increased and
chronic platelet deposition. Platelets have been implicated in
the vessel response to injury hypothesis and may participate in vessel
remodeling by release of a number of potent biological factors that can
stimulate smooth muscle cell replication, migration, matrix
production, and contraction of fibrin or collagen
gels.1 32 33 34 35 36 37 38 Platelet deposition has
previously been linked to fibrin deposition in sequential injuries.
Groves et al18 found an
50% reduction in
platelet deposition when rabbits receiving a sequential injury were
treated with heparin. In the present study, we cannot accept or
reject the role of platelets in vessel narrowing. Although
platelets were seen reacting with the luminal surface in both
FVIIai-treated and vehicle-treated vessels (Figure 5
), the study was
not designed to quantify the platelet interaction with the vessel
over the entire postoperative period. Harker et
al,39 however, have demonstrated that FVIIai
treatment significantly reduces platelet-rich thrombus deposition
at sites of carotid endarterectomy in the
baboon.
An alternative hypothesis is that luminal narrowing may involve clot retraction. Retraction in the usual sense as mediated by stimulation of platelets in a platelet-rich clot seems unlikely because of the prolonged time course of vessel narrowing. Our observation of fibrin in the vessel wall, however, raises the possibility that deposition of fibrin within the neointima may act as a scaffolding for cell migration and wound contraction. Fibrin deposition within the vessel wall may also lead to collagen synthesis and scar contracture. Although we found no large differences in histological structure of the collagen fibers, the study was not designed to assess changes in collagen structure or synthesis. Fibrin degradation products have been shown to be mitogenic for smooth muscle cells.40 Deposition of fibrin within the extracellular matrix as we have described provides increased complexity, since eutrophic remodeling may involve multiple interactions with structural proteins, resident smooth muscle cells, fibrin/fibrinogen, fibrin degradation products, and lytic enzymes.
Fibrin deposition within the vessel wall may have relevance to human restenosis. The core of the atheromatous plaque has been demonstrated to be extremely thrombogenic,41 and fibrin, fibrin degradation products, and cross-linked fibrinogen have all been found in human atherosclerotic plaques.42 43 44 45 46 47 48 Elevated plasma fibrinogen levels are associated with increased risk of both atherosclerosis49 50 and postangioplasty restenosis,51 and increases in circulating fibrinolytic parameters have been associated with atherosclerotic progression.52 53 Although the precise role of fibrin in plaque pathogenesis is yet to be determined, the balance between fibrin deposition and lysis may be intimately involved in vessel remodeling.
The role of coagulation in postangioplasty restenosis remains unclear. Inhibitors of thrombin (recombinant hirudin and hirulog) have not produced either clinical or angiographic improvement.54 55 However, the risk of bleeding complications with these compounds limits the extent of anticoagulation that can be achieved within the tissue. Because of their tissue specificity, inhibitors of tissue factor may produce more favorable results.
Our observation of extensive fibrin deposition after reinjury is
consistent with previous evidence of an increase in the ability
of the rabbit neointima to stimulate coagulation only when
reinjured.18 21 22 23 56 Groves et
al18 demonstrated the propensity for a sequential
injury, performed 1 week after the first balloon denudation to the
rabbit aorta, to stimulate mural deposition of platelet-fibrin
thrombi. Our studies suggest that the procoagulant state persists
within the neointima for at least 3 weeks after the initial
injury and that at this time the neointima promotes
extensive intramural fibrin deposition. Accumulation of tissue factor
within the neointima, as demonstrated by the OG-FVIIai
staining, suggests a causative mechanism for this procoagulant response
(Figure 7
).
Other studies have demonstrated increased tissue factorFVIIa complex activity within the artery wall after balloon injury.19 57 58 In the rat, tissue factor activity is elevated in explants from the carotid media at 1 to 2 hours after balloon injury and returns to near basal levels within 24 hours.19 Tissue factor activity has been demonstrated in the subendothelium of rabbit aortic explants,59 60 61 and luminal tissue factor activity has been shown to be elevated for 16 hours after a single balloon injury to the rabbit abdominal aorta.62 Our studies show that tissue factor protein levels in the neointima remain elevated for at least 3 weeks after a single balloon injury to the rabbit abdominal aorta. These results suggest that tissue factor accumulates and persists within the neointima but does not promote fibrin deposition until the vessel is reinjured.
The functional activity of tissue factor in vivo is controlled by a number of factors. Transport and partitioning of coagulation factors, flow-dependent enzyme kinetics, and anionic phospholipid concentrations can all play important roles.63 64 It is likely that sequential injury in our rabbit model produced cell disruption and increased permeability, which effectively altered the microenvironment within the neointima to favor coagulation. The high bolus doses of FVIIai that we applied in the acute postoperative period inhibited fibrin deposition and, by inference, tissue factor activity. Bolus doses also prolonged the partial thromboplastin times, whereas the lower maintenance doses (delivered by osmotic pump) did not. Although inhibition of systemic coagulation may not have been attained at later periods, it is possible that the low levels of circulating FVIIai did have a significant impact on maintaining inhibition of tissue factor within the vessel wall.
In summary, we have developed a model of sequential arterial injury that produces chronic inward eutrophic remodeling. The rabbit model described in the present study allows for analysis of the extrinsic pathway of coagulation on vessel wall response to injury. The first injury in this model induces a hypercoagulable state within the vessel, which may be similar to the atherosclerotic plaque. The second injury can represent interventional procedures used to treat sites of vascular stenosis. Our findings using FVIIai to inhibit tissue factor suggest that activation of the extrinsic pathway of coagulation plays a key role in the eutrophic remodeling response.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
|---|
Received September 23, 1997; accepted March 2, 1998.
| References |
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